We have constructed a cDNA library from a ras revertant cell line, in a eukaryotic expression vector and screened this library for cDNAs which are capable of suppressing ras transformation. The screening was accomplished by transfection of the cDNA library into a ras transformed cell line and selection for drug resistance and phenotypic change. Two cDNAs isolated using this strategy have been found on secondary screening to be capable of suppressing the ras transformed phenotype. The first of these cDNAs, referred to as rsp-1, is a novel gene which specifically suppresses v-Ki-ras and v-Ha-ras transformation of fibroblasts and epithelial cells. The rsp-1 protein contains a series of leucine based repeats homologous to those found in the putative ras binding region of yeast adenylyl cyclase. These findings in conjunction with the fact that it is a phylogenetically highly conserved protein suggests that rsp-1 may physically associate with ras p2l. In addition, we have identified a small RNA, 4.5S RNA, as a molecule which is capable of suppressing the ras transformed phenotype when it is expressed at a high level. High levels of 4.5S RNA are found in ras revertant cell lines and reduced levels in ras transformed cell lines compared to the level of this RNA in normal rodent fibroblasts. Our current efforts are aimed at determining the mechanisms by which these two molecules disrupt v-ras signal transduction.